【目的】验证根癌农杆菌(Agrobacterium fabrum,以前也叫Agrobacterium tumefaciens)两种储铁蛋白——饥饿细胞的DNA结合蛋白(DNA-binding protein from starved cells, Dps)和细菌铁蛋白(bacterioferritin, Bfr)的编码基因atu2477和atu...【目的】验证根癌农杆菌(Agrobacterium fabrum,以前也叫Agrobacterium tumefaciens)两种储铁蛋白——饥饿细胞的DNA结合蛋白(DNA-binding protein from starved cells, Dps)和细菌铁蛋白(bacterioferritin, Bfr)的编码基因atu2477和atu2771的功能。确定Bfr编码基因的开放阅读框。研究末端融合、血红素和个别关键氨基酸突变对Bfr功能和体外自组装的影响。探讨两种储铁蛋白的可能应用潜力。【方法】通过质粒将编码储铁蛋白的基因重新引入根癌农杆菌储铁蛋白缺失突变体中,回补储铁蛋白,验证回补的储铁蛋白编码基因是否能表达出具有储铁能力的储铁蛋白。用非变性凝胶电泳分离细胞粗提液中的蛋白质,铁特异性染色的方法鉴定电泳分离的蛋白质中是否有储铁蛋白。将不同的肽或蛋白质融合到储铁蛋白的末端,通过异源过量表达和纯化储铁蛋白的重组蛋白,用非变性凝胶电泳分析这些重组蛋白在体外的自组装。用血红素重构处理和氨基酸定点突变的方法研究血红素和个别关键氨基酸对Bfr功能和体外自组装的影响。【结果】非变性凝胶电泳和铁特异性染色结果显示,在根癌农杆菌的野生菌株、其相关突变体以及对应的回补菌株中,均仅检测到Bfr的表达,未检测到Dps的存在。当分别回补能编码161个和169个氨基酸Bfr的基因后,发现野生型菌株中的Bfr与回补编码161个氨基酸Bfr的回补菌株一样大。多肽和蛋白质的末端融合对Bfr的功能和自组装有一定影响,但不会使Bfr完全失去功能和自组装能力。结果还表明,血红素和预测可络合血红素铁的Met60的替换也只影响Bfr的功能和自组装,并未使Bfr功能完全丧失。【结论】根癌农杆菌主要通过Bfr存储铁元素。bfr基因的开放阅读框(open reading frame, ORF)以少见的UUG为起始密码子,编码产生包含161个氨基酸的蛋白质,而非169个氨基酸。根癌农杆菌的dps基因在本文的测定条件下均处于不表达状态。根癌农杆菌的Bfr和Dps蛋白均比较稳定,能够承受末端的多肽或蛋白质融合,不会使蛋白质的结构完全破坏,因此,经适当改造后具有开发应用的潜力。展开更多
The assembly of a protein complex is very important for its biological function,which can be investigated by determining the order of assembly/disassembly of its protein subunits.Although static structures of many pro...The assembly of a protein complex is very important for its biological function,which can be investigated by determining the order of assembly/disassembly of its protein subunits.Although static structures of many protein com-plexes are available in the protein data bank,their assembly/disassembly orders of subunits are largely unknown.In addi-tion to experimental techniques for studying subcomplexes in the assembly/disassembly of a protein complex,computa-tional methods can be used to predict the assembly/disassembly order.Since sampling is a nontrivial issue in simulating the assembly/disassembly process,coarse-grained simulations are more efficient than atomic simulations are.In this work,we developed computational protocols for predicting the assembly/disassembly orders of protein complexes via coarse-grained simulations.The protocols were illustrated via two protein complexes,and the predicted assembly/disassembly or-ders were consistent with the available experimental data.展开更多
Faithful transmission or restoration of epigenetic information such as repressive histone modifications through generations is crit- ical for the maintenance of cell identity. We report here that chromodomain Y-like p...Faithful transmission or restoration of epigenetic information such as repressive histone modifications through generations is crit- ical for the maintenance of cell identity. We report here that chromodomain Y-like protein （CDYL）, a chromodomain-containing transcription corepressor, is physically associated with chromatin assembly factor 1 （CAF-1） and the repiicative heUcase MCM complex. We showed that CDYL bridges CAF-1 and MCM, facilitating histone transfer and deposition during DNA replication. We demonstrated that CDYI. recruits histone-modifying enzymes G9a, SETDB1, and EZH2 to replication forks, leading to the addition of H3Kgme2/3 and H3K27me2/3 on newly deposited histone H3. Significantly, depletion of CDYL impedes early S phase progres- sion and sensitizes cells to DNA damage. Our data indicate that CDYL plays an important role in the transmission/restoration of repressive histone marks, thereby preserving the epigenetic landscape for the maintenance of cell identity.展开更多
Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins.Rec...Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins.Recently,other types of molecular constraints,especially photoresponsive linkers and functional groups,have also found increased use in a wide variety of applications.Herein,we provide a concise review of using various forms of molecular strategies to constrain proteins,thereby stabilizing their native states,gaining insight into their folding mechanisms,and/or providing a handle to trigger a conformational process of interest with light.The applications discussed here cover a wide range of topics,ranging from delineating the details of the protein folding energy landscape to controlling protein assembly and function.展开更多
Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by th...Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by this, recent chemical and biological stud- ies mainly focus on supramolecular self-assembly of proteins into high ordered architectures, especially the assembly suategy to unravel tile formation and function of protein nanostructures. In this review, we st, mmarize the progress made in the engi- neering of supramolecular protein architectures according to the strategies used to control the orient：ilion and the order of the assembly process. Furthermore, potential applications in biomedical areas of the supramolecular protein nanostructures will also be reviewed.展开更多
Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage ...Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an "affinity-guided covalent conjugation" strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative(which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made "reactive peptides" for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of "affinity-guided covalent conjugation" in various applications.展开更多
基金This work was supported by the National Key Research and Development Program of China(2021YFA1301504)the Chinese Academy of Sciences Strategic Priority Research Program(XDB37040202)the National Natural Science Foundation of China(91953101).
文摘The assembly of a protein complex is very important for its biological function,which can be investigated by determining the order of assembly/disassembly of its protein subunits.Although static structures of many protein com-plexes are available in the protein data bank,their assembly/disassembly orders of subunits are largely unknown.In addi-tion to experimental techniques for studying subcomplexes in the assembly/disassembly of a protein complex,computa-tional methods can be used to predict the assembly/disassembly order.Since sampling is a nontrivial issue in simulating the assembly/disassembly process,coarse-grained simulations are more efficient than atomic simulations are.In this work,we developed computational protocols for predicting the assembly/disassembly orders of protein complexes via coarse-grained simulations.The protocols were illustrated via two protein complexes,and the predicted assembly/disassembly or-ders were consistent with the available experimental data.
文摘Faithful transmission or restoration of epigenetic information such as repressive histone modifications through generations is crit- ical for the maintenance of cell identity. We report here that chromodomain Y-like protein （CDYL）, a chromodomain-containing transcription corepressor, is physically associated with chromatin assembly factor 1 （CAF-1） and the repiicative heUcase MCM complex. We showed that CDYL bridges CAF-1 and MCM, facilitating histone transfer and deposition during DNA replication. We demonstrated that CDYI. recruits histone-modifying enzymes G9a, SETDB1, and EZH2 to replication forks, leading to the addition of H3Kgme2/3 and H3K27me2/3 on newly deposited histone H3. Significantly, depletion of CDYL impedes early S phase progres- sion and sensitizes cells to DNA damage. Our data indicate that CDYL plays an important role in the transmission/restoration of repressive histone marks, thereby preserving the epigenetic landscape for the maintenance of cell identity.
基金supported by the National Institutes of Health(GM-065978,AG-039253)
文摘Chemical cross-linking provides an effective avenue to reduce the conformational entropy of polypeptide chains and hence has become a popular method to induce or force structural formation in peptides and proteins.Recently,other types of molecular constraints,especially photoresponsive linkers and functional groups,have also found increased use in a wide variety of applications.Herein,we provide a concise review of using various forms of molecular strategies to constrain proteins,thereby stabilizing their native states,gaining insight into their folding mechanisms,and/or providing a handle to trigger a conformational process of interest with light.The applications discussed here cover a wide range of topics,ranging from delineating the details of the protein folding energy landscape to controlling protein assembly and function.
基金supported by the National Natural Science Foundation of China(21234004,21420102007,21574056,91527302)the Chang Jiang Scholars Program of China
文摘Proteins, as the premier building blocks in nature, exhibit extraordinary ability in life activities during which process proteins mostly self-assemble into large complexes to exert prominent functions. Inspired by this, recent chemical and biological stud- ies mainly focus on supramolecular self-assembly of proteins into high ordered architectures, especially the assembly suategy to unravel tile formation and function of protein nanostructures. In this review, we st, mmarize the progress made in the engi- neering of supramolecular protein architectures according to the strategies used to control the orient：ilion and the order of the assembly process. Furthermore, potential applications in biomedical areas of the supramolecular protein nanostructures will also be reviewed.
基金supported by the University Grants Committee of Hong Kong (ECS grant CUHK 404812, GRF grants 403711 and 404413, and Ao E/M-09/12)
文摘Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an "affinity-guided covalent conjugation" strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative(which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made "reactive peptides" for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of "affinity-guided covalent conjugation" in various applications.
